Drug-Resistant Epilepsy in Tuberous Sclerosis Complex Is Associated With TSC2 Genotype: More Findings From the Preventing Epilepsy Using Vigatrin (PREVeNT) Trial.

BACKGROUND: Children with tuberous sclerosis complex (TSC) are at high risk for drug-resistant epilepsy (DRE). The ability to stratify those at highest risk for DRE is important for counseling and prompt, aggressive management, necessary to optimize neurocognitive outcomes. Using the extensively phenotyped PREVeNT cohort, we aimed to characterize whether the TSC genotype was associated with DRE. METHODS: The study group (N = 70) comprised participants with TSC enrolled at age less than or equal to six months with detailed epilepsy and other phenotypic and genotypic data, prospectively collected as part of the PREVeNT trial. Genotype-phenotype correlations of DRE, time to first abnormal electroencephalography, and time to epilepsy onset were compared using Fisher exact test and regression models. RESULTS: Presence of a TSC2 pathogenic variant was significantly associated with DRE, compared with TSC1 and participants with no pathogenic mutation identified. In fact, all participants with DRE had a TSC2 pathogenic variant. Furthermore, TSC2 variants expected to result in no protein product were associated with higher risk for DRE. Finally, TSC1 pathogenic variants were associated with later-onset epilepsy, on average 21.2 months later than those with other genotypes. CONCLUSIONS: Using a comprehensively phenotyped cohort followed from infancy, this study is the first to delineate genotype-phenotype correlations for epilepsy severity and onset in children with TSC. Patients with TSC2 pathogenic variants, especially TSC2 pathogenic variants predicted to result in lack of TSC2 protein, are at highest risk for DRE, and are likely to have earlier epilepsy onset than those with TSC1. Clinically, these insights can inform counseling, surveillance, and management.

miR-27a-3p promotes inflammatory response in infectious endophthalmitis via targeting TSC1.

Infectious endophthalmitis (IE) poses a significant threat to vision. This study aimed to explore the impact of microRNA (miR)-27a-3p on inflammation in IE. A rat model was developed through intravitreal injection of lipopolysaccharide. Clinical and demographic data were collected for 54 participants: 31 diagnosed with IE and 23 non-infectious patients with idiopathic macular holes. Expression levels of miR-27a-3p and inflammatory genes were quantified via reverse transcription quantitative polymerase chain reaction. Concentrations of inflammatory cytokines in human vitreous samples were measured using enzyme-linked immunosorbent assay. In vitro studies were conducted to explore the target gene of miR-27a-3p. The final animal experiments further verified the role of miR-27a-3p and tuberous sclerosis complex (TSC)1 in inflammatory responses. Results showed that miR-27a-3p was elevated in LPS-treated rats and IE patients. Thirty-one IE patients were divided into the High (n = 15) and Low (n = 16) groups according to the expression of miR-27a-3p. No significant differences were observed in baseline clinical and demographic characteristics between the control and IE patient groups. Pro-inflammatory cytokine mRNA levels and concentrations were notably increased in both LPS-treated rats and the High group of patients. Besides, results showed that TSC1 is a target gene of miR-27a-3p. Moreover, TSC1 inhibition promoted inflammation in rat vitreous samples. In summary, our findings suggested that miR-27a-3p exacerbated inflammatory responses in IE though targeting TSC1, offering novel insights for potential therapeutic strategies targeting miR-27a-3p in the clinical management of IE.

Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-ß RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-ß RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-ß RII-inactivated cancers.

tRNA m1A modification regulate HSC maintenance and self-renewal via mTORC1 signaling.

Haematopoietic stem cells (HSCs) possess unique physiological adaptations to sustain blood cell production and cope with stress responses throughout life. To maintain these adaptations, HSCs rely on maintaining a tightly controlled protein translation rate. However, the mechanism of how HSCs regulate protein translation remains to be fully elucidated. In this study, we investigate the role of transfer RNA (tRNA) m1A58 'writer' proteins TRMT6 and TRMT61A in regulating HSCs function. Trmt6 deletion promoted HSC proliferation through aberrant activation of mTORC1 signaling. TRMT6-deficient HSCs exhibited an impaired self-renewal ability in competitive transplantation assay. Mechanistically, single cell RNA-seq analysis reveals that the mTORC1 signaling pathway is highly upregulated in HSC-enriched cell populations after Trmt6 deletion. m1A-tRNA-seq and Western blot analysis suggest that TRMT6 promotes methylation modification of specific tRNA and expression of TSC1, fine-tuning mTORC1 signaling levels. Furthermore, Pharmacological inhibition of the mTORC1 pathway rescued functional defect in TRMT6-deficient HSCs. To our knowledge, this study is the first to elucidate a mechanism by which TRMT6-TRMT61A complex-mediated tRNA-m1A58 modification regulates HSC homeostasis.

Tuberous Sclerosis Complex and the kidneys: what nephrologists need to know.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the development of hamartomas in the central nervous system, heart, skin, lungs, and kidneys and other manifestations including seizures, cortical tubers, radial migration lines, autism and cognitive disability. The disease is associated with pathogenic variants in the TSC1 or TSC2 genes, resulting in the hyperactivation of the mTOR pathway, a key regulator of cell growth and metabolism. Consequently, the hyperactivation of the mTOR pathway leads to abnormal tissue proliferation and the development of solid tumors. Kidney involvement in TSC is characterized by the development of cystic lesions, renal cell carcinoma and renal angiomyolipomas, which may progress and cause pain, bleeding, and loss of kidney function. Over the past years, there has been a notable shift in the therapeutic approach to TSC, particularly in addressing renal manifestations. mTOR inhibitors have emerged as the primary therapeutic option, whereas surgical interventions like nephrectomy and embolization being reserved primarily for complications unresponsive to clinical treatment, such as severe renal hemorrhage. This review focuses on the main clinical characteristics of TSC, the mechanisms underlying kidney involvement, the recent advances in therapy for kidney lesions, and the future perspectives.

Modeling mTORopathy-related epilepsy in cultured murine hippocampal neurons using the multi-electrode array.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a ubiquitous cellular pathway. mTORopathies, a group of disorders characterized by hyperactivity of the mTORC1 pathway, illustrate the prominent role of the mTOR pathway in disease pathology, often profoundly affecting the central nervous system. One of the most debilitating symptoms of mTORopathies is drug-resistant epilepsy, emphasizing the urgent need for a deeper understanding of disease mechanisms to develop novel anti-epileptic drugs. In this study, we explored the multiwell Multi-electrode array (MEA) system as a tool to identify robust network activity parameters in an approach to model mTORopathy-related epilepsy in vitro. To this extent, we cultured mouse primary hippocampal neurons on the multiwell MEA to identify robust network activity phenotypes in mTORC1-hyperactive neuronal networks. mTOR-hyperactivity was induced either through deletion of Tsc1 or overexpression of a constitutively active RHEB variant identified in patients, RHEBp.P37L. mTORC1 dependency of the phenotypes was assessed using rapamycin, and vigabatrin was applied to treat epilepsy-like phenotypes. We show that hyperactivity of the mTORC1 pathway leads to aberrant network activity. In both the Tsc1-KO and RHEB-p.P37L models, we identified changes in network synchronicity, rhythmicity, and burst characteristics. The presence of these phenotypes is prevented upon early treatment with the mTORC1-inhibitor rapamycin. Application of rapamycin in mature neuronal cultures could only partially rescue the network activity phenotypes. Additionally, treatment with the anti-epileptic drug vigabatrin reduced network activity and restored burst characteristics. Taken together, we showed that mTORC1-hyperactive neuronal cultures on the multiwell MEA system present reliable network activity phenotypes that can be used as an assay to explore the potency of new drug treatments targeting epilepsy in mTORopathy patients and may give more insights into the pathophysiological mechanisms underlying epilepsy in these patients.

CDK4/6 inhibitors dephosphorylate RNF26 to stabilize TSC1 and increase the sensitivity of ccRCC to mTOR inhibitors.

BACKGROUND: The combined use of CDK4/6 inhibitors and mTOR inhibitors has achieved some clinical success in ccRCC. Exploring the underlying mechanism of the CDK4/6 pathway in cancer cells and the drug interactions of CDK4/6 inhibitors in combination therapy could help identify new therapeutic strategies for ccRCC. Notably, CDK4/6 inhibitors inactivate the mTOR pathway by increasing the protein levels of TSC1, but the mechanism by which CDK4/6 inhibitors regulate TSC1 is still unclear. METHODS: Mass spectrometry analysis, coimmunoprecipitation analysis, GST pull-down assays, immunofluorescence assays, Western blot analysis and RT‒qPCR analysis were applied to explore the relationships among CDK4, RNF26 and TSC1. Transwell assays, tube formation assays, CCK-8 assays, colony formation assays and xenograft assays were performed to examine the biological role of RNF26 in renal cancer cells.TCGA-KIRC dataset analysis and RT‒qPCR analysis were used to examine the pathways affected by RNF26 silencing. RESULTS: CDK4/6 inhibitors stabilized TSC1 in cancer cells. We showed that CDK4 enhances the interaction between TSC1 and RNF26 and that RNF26 activates the mTOR signaling pathway in ccRCC, contributes to ccRCC progression and angiogenesis, and promotes tumorigenesis. We then found that RNF26 functions as an E3 ligase of TSC1 to regulate CDK4-induced TSC1. This finding suggested that RNF26 promotes ccRCC progression and angiogenesis to some extent by negatively regulating TSC1. CONCLUSION: Our results revealed a novel CDK4/RNF26/TSC1 axis that regulates the anticancer efficacy of CDK4/6 inhibitors and mTOR inhibitors in ccRCC.

The extent of kidney involvement in paediatric tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis (TSC)-associated kidney disease is a leading cause of mortality in adults with TSC. This study aimed to understand TSC features in children, particularly kidney involvement, to inform clinical care for this specific group. METHODS: This retrospective cohort study included all paediatric (< 19 years) TSC cases at a large tertiary paediatric nephrology centre. Relevant data were collected from patients' records, statistical analyses were performed to identify associations between variables, survival probabilities were estimated with Kaplan‒Meier curves, and log-rank tests were conducted to assess survival differences among genetic mutations. RESULTS: A total of 182 children with TSC were included. Among the 145 children with available kidney imaging data, 78.6% (114/145) exhibited kidney lesions. Angiomyolipomas (AMLs) were significantly more prevalent in the TSC2 mutation group (p = 0.018). Children with TSC2 mutations generally had poorer lesion-free survival than those with TSC1 mutations, but this difference was only significant for AMLs (p = 0.030). The change in size of largest AMLs increased with age and doubled in children above 9 years; a similar pattern was observed when stratified by genetic mutation. In contrast, kidney cysts exhibited two peaks: one in children under 5 years (2.31 mm/year) and the second in children between 15-19 years (2.82 mm/year). Chronic kidney disease was observed in 12.3% (10/81) of children, and high-risk AMLs above 3 cm were observed in 9% (13/145). CONCLUSIONS: While TSC kidney disease emerges later in the disease course than neurological features, our findings emphasise the importance of kidney surveillance during childhood, including routine kidney imaging, kidney function, and blood pressure monitoring.

Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

Malignant tumors in tuberous sclerosis complex: a case report and review of the literature.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare, autosomal dominant genetic disease that arises from TSC1 or TSC2 genetic mutations. These genetic mutations can induce the development of benign tumors in any organ system with significant clinical implications in morbidity and mortality. In rare instances, patients with TSC can have malignant tumors, including renal cell carcinoma (RCC) and pancreatic neuroendocrine tumor (PNET). It is considered a hereditary renal cancer syndrome despite the low incidence of RCC in TSC patients. TSC is typically diagnosed in prenatal and pediatric patients and frequently associated with neurocognitive disorders and seizures, which are often experienced early in life. However, penetrance and expressivity of TSC mutations are highly variable. Herein, we present a case report, with associated literature, to highlight that there exist undiagnosed adult patients with less penetrant features, whose clinical presentation may contain non-classical signs and symptoms, who have pathogenic TSC mutations. CASE PRESENTATION: A 31-year-old female with past medical history of leiomyomas status post myomectomy presented to the emergency department for a hemorrhagic adnexal cyst. Imaging incidentally identified a renal mass suspicious for RCC. Out of concern for hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, the mass was surgically removed and confirmed as RCC. Discussion with medical genetics ascertained a family history of kidney cancer and nephrectomy procedures and a patient history of ungual fibromas on the toes. Genetic testing for hereditary kidney cancer revealed a 5'UTR deletion in the TSC1 gene, leading to a diagnosis of TSC. Following the diagnosis, dermatology found benign skin findings consistent with TSC. About six months after the incidental finding of RCC, a PNET in the pancreatic body/tail was incidentally found on chest CT imaging, which was removed and determined to be a well-differentiated PNET. Later, a brain MRI revealed two small cortical tubers, one in each frontal lobe, that were asymptomatic; the patient's history and family history did not contain seizures or learning delays. The patient presently shows no evidence of recurrence or metastatic disease, and no additional malignant tumors have been identified. CONCLUSIONS: To our knowledge, this is the first report in the literature of a TSC patient without a history of neurocognitive disorders with RCC and PNET, both independently rare occurrences in TSC. The patient had a strong family history of renal disease, including RCC, and had several other clinical manifestations of TSC, including skin and brain findings. The incidental finding and surgical removal of RCC prompted the genetic evaluation and diagnosis of TSC, leading to a comparably late diagnosis for this patient. Reporting the broad spectrum of disease for TSC, including more malignant phenotypes such as the one seen in our patient, can help healthcare providers better identify patients who need genetic evaluation and additional medical care.

Review of the spectrum of tuberous sclerosis complex: The Saudi Arabian Experience.

OBJECTIVES: To determine the prevalence of tuberous sclerosis complex (TSC) in the paediatric Saudi population and to characterise the range of clinical symptoms, neurocutaneous findings, neuroimaging results, and complications of the disease. METHODS: A total of 61 genetically confirmed TSC patients from the National Guard Health Affairs (NGHA) in Saudi Arabia were the subject of this retrospective descriptive analysis. The data were presented using descriptive measures. RESULTS: The mean age at diagnosis was found to be 4.9 years. Subependymal nodules (86.9%), numerous cortical tubers and/or radial migration lines (63.9%), and hypomelanotic macules (63.9%) were the 3 most common significant criteria. The vast majority (86.9%) of those diagnosed had epilepsy, of which 50% were considered medically intractable. Nearly half of our subjects underwent genetic testing, which revealed that TSC2 predominated over TSC1. Symptoms of Tuberous Sclerosis Complex-Associated Neuropsychiatric Disorders (TAND) were present in 66.7% of TSC1 patients and 73.9% of TSC2 patients. CONCLUSION: The findings of this study demonstrate that the clinical spectrum of TSC among Saudi children is consistent with the body of existing literature. The TSC2 was more prevalent than TSC1. The most frequent signs were cutaneous and neurological. Monitoring TSC patients regularly is crucial to identify any issues as soon as possible.

The autophagy-mediated mechanism via TSC1/mTOR signaling pathway in thiram-induced tibial dyschondroplasia of broilers.

Thiram is a member of the dithiocarbamate family and is widely used in agriculture, especially in low-income countries. Its residues lead to various diseases, among which tibial dyschondroplasia (TD) in broiler chickens is the most common. Recent studies have also demonstrated that thiram residues may harm human health. Our previous study showed that the activity of the mTOR (mammalian target of rapamycin) signaling pathway has changed after thiram exposure. In the current study, we investigated the effect of autophagy via the mTOR signaling pathway after thiram exposure in vitro and in vivo. Our results showed that thiram inhibited the protein expression of mTOR signaling pathway-related genes such as p-4EBP1 and p-S6K1. The analysis showed a significant increase in the expression of key autophagy-related proteins, including LC3, ULK1, ATG5, and Beclin1. Further investigation proved that the effects of thiram were mediated through the downregulation of mTOR. The mTOR agonist MHY-1485 reverse the upregulation of autophagy caused by thiram in vitro. Moreover, our experiment using knockdown of TSC1 resulted in chondrocytes expressing lower levels of autophagy. In conclusion, our results demonstrate that thiram promotes autophagy via the mTOR signaling pathway in chondrogenesis, providing a potential pharmacological target for the prevention of TD.

A dermatological assessment of pediatric patients with tuberous sclerosis complex (TSC).

BACKGROUND: Tuberous sclerosis complex (TSC) is a multisystem neurocutaneous syndrome with variable phenotypes. Recent updates of TSC diagnostic criteria reaffirmed the defined genetic diagnostic criterion as the finding of a pathogenic DNA alteration in either TSC1 or TSC2 genes. It also slightly modified definite clinical diagnostic criteria. TSC-associated skin lesions in infancy are important clinical signs to select individuals with possible TSC for a closer clinical follow-up and genetic testing. OBJECTIVE: To raise awareness of the updated TSC diagnosis criteria; to assess the frequency of skin lesions in TSC patients as well as the first dermatological presentation; and to associate the findings with either TSC1 or TSC2 mutations. METHODS: Observational cross-sectional study. Clinical and genetic data were retrospectively collected from 37 TSC patients from a Brazilian University Hospital. Patients with skin signs were examined and prospectively assessed for 12 months. RESULTS: The earliest cutaneous lesions were hypomelanotic macules, which together with angiofibromas were the most frequent dermatological lesions. The total pathogenic DNA alteration ratio between TSC2 and TSC1 genes was 8:1. The frequency of a TSC2 pathogenic variant was 10-fold greater in the presence of ungual fibromas. STUDY LIMITATIONS: Small sample and a limited number of patients with TSC1 pathogenic variants. CONCLUSION: Clinicians should be knowledgeable about TSC updated diagnostic criteria. Patients need to be followed up by a multidisciplinary team and treated accordingly. Early detection of cutaneous lesions is important for TSC diagnosis. A significant association between TSC2 gene pathogenic alterations and ungual fibromas is described.

Research of the unrecognised functions of miR-375 in prostate cancer cells.

Many cancers, including prostate cancer, have miRNAs with altered expression levels. These miRNAs play a pivotal role in regulating cancer initiation, invasion, and metastasis. miRNAs are an important component in cancer diagnosis and therapy and can play a key role as biomarkers or chemotherapeutic agents.  This investigation aimed to show the effects of miR-375 on PCa. In this project, target prediction tools and the KEGG pathway were performed to determine the potential targets of miR-375. Transfection was performed using miR-375 mimic and inhibitor. The actions of miRNAs on cell viability and migration were examined in PCa cells. In addition, qRT-PCR was executed to evaluate changes in gene expression in the PI3K-mTOR pathway. The analyses exposed that the upregulation of miR-375 repressed the viability at 48 h. While stimulation of miR-375 did not repress the migration, suppression of miR-375 reduced the migration at 24 and 48 hours. The predicted target TSC1 gene is not directly targeted by miR-375. Interestingly, in response to PIK3CA increase, mTOR expression was suppressed in all cells except LNCAP cells. In conclusion, miR-375 has anti-proliferative and cell migration inhibitory effects in prostate cancer. However, studies demonstrate that miR-375 may have tumor suppressor and oncogenic effects when considering cell molecular differences.

Recurrent Tuberous Sclerosis Complex/Mammalian Target of Rapamycin Mutations Define Primary Renal Hemangioblastoma as a Unique Entity Distinct From Its Central Nervous System Counterpart.

ABSTRACT: Renal hemangioblastoma (HB) is a rare subset of HBs arising outside of the central nervous system (CNS), with its molecular drivers remaining entirely unknown. There were no significant alterations detected in previous studies, including von Hippel-Lindau gene alterations, which are commonly associated with CNS-HB. This study aimed to determine the real molecular identity of renal HB and better understand its relationship with CNS-HB. A cohort of 10 renal HBs was submitted for next-generation sequencing technology. As a control, 5 classic CNS-HBs were similarly analyzed. Based on the molecular results, glycoprotein nonmetastatic B (GPNMB) immunohistochemistry was further performed in the cases of renal HB and CNS-HB. Mutational analysis demonstrated that all 10 renal HBs harbored somatic mutations in tuberous sclerosis complex 1 ( TSC1 , 5 cases), TSC2 (3 cases), and mammalian target of rapamycin (2 cases), with the majority classified as pathogenic or likely pathogenic. The CNS-HB cohort uniformly demonstrated somatic mutations in the von Hippel-Lindau gene. GPNMB was strong and diffuse in all 10 renal HBs and completely negative in CNS-HBs, reinforcing the molecular findings. Our study reveals a specific molecular hallmark in renal HB, characterized by recurrent TSC/mammalian target of rapamycin mutations, which defines it as a unique entity distinct from CNS-HB. This molecular finding potentially expands the therapeutic options for patients with renal HB. GPNMB can be considered for inclusion in immunohistochemical panels to improve renal HB identification.

Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection.

Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.

A large deletion in TSC2 causes tuberous sclerosis complex by dysregulating PI3K/AKT/mTOR signaling pathway.

BACKGROUND/AIM: Tuberous sclerosis complex (TSC) is a multi-system syndrome caused by loss-of-function mutation in TSC1 or TSC2. Most TSC patients present with cardiac rhabdomyoma or cortical tubers during fetal life, and the symptoms are not uniform as their age. The gene products of TSC1/2 are components of the TSC protein complex and are important role in the PI3K/AKT/mTOR (PAM) signaling pathway. Based on three members of a family with variable expressivity, the purpose of this study was to clarify the clinical features of TSC in different age groups and to analyze the genetic characteristics of TSC2 gene. METHODS: Clinical exome sequencing and co-segregation were used to identify a three-generation family with four affected individuals. HEK-293T cell model was constructed for subsequent experiments. Quantitative RT-PCR, western blotting, and subcellular localization were used to analyze the expression effect of TSC2 mutation. CCK-8 assay, wound healing assay, and cell cycle analysis were used to analyze the function effect of TSC2 mutation. RESULT: We identified a TSC family with heterozygous deletion of exon 4 in TSC2 by clinical exon sequencing. Sanger sequencing indicated that the affected individuals have 2541-bp deletion that encompassed exon 4 and adjacent introns. Deletion of exon 4 decreased the TSC2 mRNA and protein levels in HEK-293T cells, and activated the PI3K/AKT/mTOR pathway, thereby altering the cell cycle and promoting cell proliferation and migration. CONCLUSION: We confirmed the pathogenicity of the large deletion in TSC2 in a three- generations family.. Deletion of exon 4 of TSC2 affected cell proliferation, migration, and cell cycle via abnormal activation of the PAM pathway. This study evaluated the pathogenic effect of deletion of exon 4 of TSC2 and investigated the underlying mechanism.

The role of TSC1 and TSC2 proteins in neuronal axons.

Tuberous Sclerosis Complex 1 and 2 proteins, TSC1 and TSC2 respectively, participate in a multiprotein complex with a crucial role for the proper development and function of the nervous system. This complex primarily acts as an inhibitor of the mechanistic target of rapamycin (mTOR) kinase, and mutations in either TSC1 or TSC2 cause a neurodevelopmental disorder called Tuberous Sclerosis Complex (TSC). Neurological manifestations of TSC include brain lesions, epilepsy, autism, and intellectual disability. On the cellular level, the TSC/mTOR signaling axis regulates multiple anabolic and catabolic processes, but it is not clear how these processes contribute to specific neurologic phenotypes. Hence, several studies have aimed to elucidate the role of this signaling pathway in neurons. Of particular interest are axons, as axonal defects are associated with severe neurocognitive impairments. Here, we review findings regarding the role of the TSC1/2 protein complex in axons. Specifically, we will discuss how TSC1/2 canonical and non-canonical functions contribute to the formation and integrity of axonal structure and function.

Identification of a complex intrachromosomal inverted insertion in the long arm of chromosome 9 as a cause of tuberous sclerosis complex in a Korean family.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder, caused by a loss-of-function of either TSC1 or TSC2 gene. However, in 10%-15% TSC patients there is no pathogenic variant identified in either TSC1 or TSC2 genes based on standard clinical testing. METHODS: In this study, genome sequencing was performed for families with clinical diagnosis of TSC with negative results from TSC1 and TSC2 single-gene tests. RESULTS: Herein, we report a family presenting a classical TSC phenotype with an unusual, complex structural variant involving the TSC1 gene: an intrachromosomal inverted insertion in the long arm of chromosome 9. We speculate that the inverted 9q33.3q34.13 region was inserted into the q31.2 region with the 3'-end of the breakpoint of the inversion being located within the TSC1 gene, resulting in premature termination of TSC1. CONCLUSIONS: In this study, we demonstrate the utility of genome sequencing for the identification of complex chromosomal rearrangement. Because the breakpoints are located within the deep intronic/intergenic region, this copy-neutral variant was missed by the TSC1 and TSC2 single-gene tests and contributed to an unknown etiology. Together, this finding suggests that complex structural variants may be underestimated causes for the etiology of TSC.